Apoptosis, or programmed cell death, is a naturally occurring process that has been strongly conserved during evolution to prevent uncontrolled cell proliferation. This form of cell suicide plays a crucial role in ensuring the development and maintenance of multicellular organisms by eliminating superfluous or unwanted cells. However, if this process becomes overstimulated, cell loss and degenerative disorders including neurological disorders such as Alzheimers, Parkinsons, ALS, retinitis pigmentosa and blood cell disorders can result. Stimuli which can trigger apoptosis include growth factors such as tumor necrosis factor (TNF), Fas and transforming growth factor beta (TGF ), neurotransmitters, growth factor withdrawal, loss of extracellular matrix attachment and extreme fluctuations in intracellular calcium levels (Afford and Randhawa, Mol. Pathol., 2000, 53, 55-63).
Alternatively, insufficient apoptosis, triggered by growth factors, extracellular matrix changes, CD40 ligand, viral gene products neutral amino acids, zinc, estrogen and androgens, can contribute to the development of cancer, autoimmune disorders and viral infections (Afford and Randhawa, Mol. Pathol., 2000, 53, 55-63). Consequently, apoptosis is regulated under normal circumstances by the interaction of gene products that either induce or inhibit cell death and several gene products which modulate the apoptotic process have now been identified.
Apoptosis manifests itself in two major downstream execution programs: the caspase pathway and mitochondrial dysfunction (Korsmeyer et al., Cold Spring Harb. Symp. Quant. Biol., 1999, 64, 343-350; Korsmeyer et al., Cell Death Differ., 2000, 7, 1166-1173). The BCL-2 family members play a pivotal role in determining whether a cell will live or die by acting at the mitochondrial level. This family includes both pro-apoptotic and anti-apoptotic proteins and the ratio between these subsets is a determinant of the susceptibility of the cells to death signals (Korsmeyer et al., Cold Spring Harb. Symp. Quant. Biol., 1999, 64, 343-350). Members of the BCL-2 family have the ability to form homodimers or heterodimers, suggesting neutralizing competition among these proteins. An additional characteristic of functional significance is the ability of BCL-2 family members to act as integral membrane proteins (Korsmeyer et al., Cold Spring Harb. Symp. Quant. Biol., 1999, 64, 343-350).
BCL2-associated X protein (also known as BAX), is a pro-apoptotic BCL-2 family member. Activation of BCL2-associated X protein involves subcellular translocation and dimerization. In viable cells, a substantial portion of BCL2-associated X protein is monomeric and found either in the cytosol or loosely associated with membranes. Following a death stimulus, cytosolic monomeric BCL2-associated X protein translocates to the mitochondria where it becomes a cross-linkable, integral membrane protein. The ability of BCL2-associated X protein to form distinct ion-conductive membrane pores may be, in part, responsible for mitochondrial dysfunction that leads to cell death (Korsmeyer et al., Cold Spring Harb. Symp. Quant. Biol., 1999, 64, 343-350; Korsmeyer et al., Cell Death Differ., 2000, 7, 1166-1173).
BCL2-associated X protein has been cloned (Oltvai et al., Cell, 1993, 74, 609-619) and mapped to chromosome 19q13.3 (Apte et al., Genomics, 1995, 26, 592-594; Chou et al., Cancer Genet. Cytogenet., 1996, 88, 136-140), and has been shown to encode a number of splice variants, including BAX-alpha, BAX-beta, BAX-gamma (Oltvai et al., Cell, 1993, 74, 609-619), BAX-delta (Apte et al., Genomics, 1995, 26, 592-594), BAX-omega (Zhou et al., J. Biol. Chem., 1998, 273, 11930-11936) and BAX-epsilon (Shi et al., Biochem. Biophys. Res. Commun., 1999, 254, 779-785.). Nucleotide sequences encoding BAX-alpha, BAX-beta and BAX-gamma are disclosed and claimed in U.S. Pat. Nos. 5,691,179 and 5,955,595 (Korsmeyer, 1997; Korsmeyer, 1999). Nucleotide sequences encoding BAX-omega are disclosed and claimed in U.S. Pat. No. 6,140,484 and corresponding PCT publication WO 97/01635 (Bitler et al., 2000; Bitler et al., 1997). Also disclosed in U.S. Pat. No. 6,140,484 is a 22-mer antisense oligonucleotide directed against the exon5/intron5 junction of human BAX-omega (Bitler et al., 2000).
Overexpression of BCL2-associated X protein has been observed in human diseases including, glomerular disease (Yoshimura et al., Nephrol. Dial. Transplant, 1999, 14, 55-57), Hodgkin's disease (Brousset et al., Blood, 1996, 87, 2470-2475), cartilage-hair hypoplasia (Yel et al., J. Clin. Immunol., 1999, 19, 428-434) and ocular complications arising from diabetes (Podesta et al., Am. J. Pathol., 2000, 156, 1025-1032). In addition, animal models of human disease have been used to determine that overexpression of BCL2-associated X protein also may occur in Alzheimer's disease (MacGibbon et al., Brain Res., 1997, 750, 223-234), Parkinson's disease (Vila et al., Proc. Natl. Acad. Sci. U.S.A., 2001, 98, 2837-2842), familial amyotrophic lateral sclerosis (Vukosavic et al., J. Neurochem., 1999, 73, 2460-2468) and scrapie infections (Park et al., NeuroReport, 2000, 11, 1677-1682).
BCL2-associated X protein knockout mice have been generated and proved viable, but displayed lineage-specific aberrations in cell death (Knudson et al., Science, 1995, 270, 96-99). Additionally, prolongation of ovarian life span into advanced chronological age has been observed in BCL2-associated X protein knockout mice (Perez et al., Nat. Genet., 1999, 21, 200-203).
Currently there exists a need to identify methods of modulating apoptosis for the therapeutic treatment of human diseases.
Antisense oligonucleotides, 20 nucleotides in length, targeting bases 83-102 and 103-122 of human BCL2-associated X protein have been used to inhibit BCL2-associated X protein in human myeloid cells (Manfredini et al., Antisense Nucleic Acid Drug Dev., 1998, 8, 341-350) and neutrophils (Dibbert et al., Proc. Natl. Acad. Sci. U.S.A., 1999, 96, 13330-13335).
Currently, there remains a need for additional agents capable of effectively and selectively inhibiting the expression of BCL2-associated X protein.
Modified antisense phosphorothioate oligonucleotides and unmodified oligodeoxynucleotides targeting the start codon of rat BCL2-associated X protein have been used to inhibit BCL2-associated X protein in rat neurons (Gillardon et al., J. Neurosci. Res., 1996, 43, 726-734; Isenmann et al., Cell Death Differ., 1999, 6, 673-682; Tamatani et al., J. Neurochem., 1998, 71, 1588-1596; Tamatani et al., Cell Death Differ., 1998, 5, 911-919) and R6 fibroblasts (Otter et al., J. Biol. Chem., 1998, 273, 6110-6120).
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of BCL2-associated X protein gene expression and cellular processes.
The present invention provides compositions and methods for modulating BCL2-associated X protein expression, including modulation of splice variants of BCL2-associated X protein including BAX-alpha, BAX-beta, BAX-gamma, BAX-delta, BAX-omega, and BAX-epsilon.